Project Summary/Abstract Mucosal healing is considered an important clinical end-point of gastrointestinal disorders, including inflammatory bowel diseases (IBD). As such, it is an attractive target for developing therapies aimed at achieving sustained clinical remission. PMN presence in the intestinal mucosa is often associated with disease severity and tissue damage, however, PMN contributions to resolution of inflammation are increasingly recognized. Thus, while our view of PMNs as terminally differentiated phagocytes that promote tissue damage is changing, mechanisms underlying beneficial roles of PMNs remain poorly understood. Likewise, although macrophage (M?) contributions to mounting immune responses in the gut and to resolution of inflammation are well recognized, molecular cues that instruct their effector functions remain to be defined. Our ongoing studies indicate a novel and beneficial contribution of tissue PMNs to the resolution of inflammation and mucosal injury. We have identified several novel mechanisms and new molecular players by which PMNs instruct the activity of inflammatory gut M?s to promote mucosal healing. We found that PMN extracellular vesicles (EVs) mediate localized transfer of regulatory micro-RNAs (miRNAs) and pro-repair factors (e.g. TGF- ?1), which respectively, through parallel activity suppress inflammatory and promote pro-repair cytokine expression in gut M?s. We also identified a new regulatory role for a well-characterized PMN adhesion ligand, ICAM-1, in regulating M? efferocytotic activity. ICAM-1 is highly upregulated during M? activation (its expression is restricted to inflammatory M?s) and when ligated by PMN binding, critically regulates clearance of apoptotic/necrotic epithelial cells in injured tissue. Therefore, the overall goal of this proposal is to test a novel concept whereby tissue infiltrating PMNs facilitate reprogramming of gut M?s to promote inflammatory resolution of the injured intestinal mucosa. Proposed experiments will use state of the art imaging techniques, powerful in vivo injury models combined with innovative molecular and biochemical approaches to: 1. Determine how PMN-M? interactions facilitate injury resolution in the intestinal mucosa. 2. Determine how PMN-EVs enhance the pro-repair activity of inflammatory wound M?s and 3. Determine how PMN binding and ICAM-1 signaling in wound M?s regulate efferocytosis and facilitate wound debridement. Our studies will define new mechanisms governing innate immune cell interactions in wounded mucosa and their function in injury resolution.